In the face of escalating oil prices and the growing environmental concerns over the use and depletion of non-renewable energy resources, there is a growing desire to reduce this dependency, stimulating interest in the use of fermentation processes for the large-scale production of alternative biofuels such as ethanol (Editorial (July, 2006) Bioethanol needs biotech now. Nat Biotechnol. 24, 725, Gray, K A et al. (2006) Bioethanol. Cur. Opin. Chem. Biol. 10, 141-6). As a fuel, ethanol is mainly of interest as a petrol additive, or substitute, because ethanol-blended fuel produces a cleaner, more complete combustion that reduces green-house gas and toxic emissions. The production of ethanol in the US has increased from less than 800 million liters in 1980 to about 19 billion liters in 2006, but there are plans to boost production to more than 36 billion liters by 2012. In 2004 this industry added U$14 billions to the US Gross Domestic Product (Securing America's energy future (2006), brochure produced by the Renewable Fuels Association). However, the US is not the largest producer of bioethanol, this occurs in Brazil. Recently, Brazil and the UK have agreed to co-operate in the development of facilities for bioethanol production in other countries, such as South Africa.
As a consequence of the surge in demand for biofuels, ethanol-producing microorganisms, such as the Gram-negative bacterium Zymomonas mobilis, are of considerable interest due to their potential for the production of bioethanol (Jeffries, T W (2005) Ethanol fermentation on the move. Nat Biotechnol. 23, 40-1). Z. mobilis attracted attention early in the development of ethanol fuel technology because it grows and ferments rapidly, and has a product rate and yield significantly higher than that of yeast. Furthermore, it tolerates high levels of ethanol, a virtually unique property among bacteria, and sugars. It is distinctive in that it uses the Entner-Doudoroff (E-D) pathway for glucose metabolism rather than the more familiar glycolytic pathway used by most bacteria and yeast. The key enzyme of the E-D pathway is pyruvate decarboxylase (PDC), which is only rarely found in bacteria. Unlike glycolysis, which can theoretically generate two moles of ATP for each mole of glucose fermented to ethanol, the E-D pathway has a net yield of only one ATP per mole of glucose. This low yield of ATP results in low cell mass and allows higher ethanol yields. Although wild-type strains of Z. mobilis can only use glucose, fructose and sucrose as carbon substrates, recent research has focused on the development of recombinant strains capable of converting cheaper lignocellulosic hydrolysates to ethanol. All in, these features of Z. mobilis have made it an attractive candidate for bioethanol production (Seo, J-S et al. (2005) The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol. 23, 63-8).
To keep in step with the growing demand for biofuels will require the engineering of new strains of fermentative microorganisms that can produce these more efficiently; and to enable the engineering of such strains will require more detailed information on the genetic circuits involved in regulating biofuel production.
U.S. Pat. No. 5,514,583 discloses a transformed Z. mobilis xylose fermenting strain (CP4/pZB4 and pZB5) having exogenous genes, and plasmid vectors (pZB4 and pZB5) encoding xylose isomerase, xylulokinase, transaldolase and transketolase, and further comprising at least one promoter (Pgap and Peno) recognized by Zymomonas which regulates the expression of at least one of said genes. The microorganism is capable of growing on xylose as a sole carbon source, and fermenting xylose to ethanol at about 88% of the maximum theoretic yield.
U.S. Pat. Nos. 5,712,133 and 5,726,053 relates to, inter alia, Z. mobilis arabinose fermenting transformants (39676/pZB 206), containing exogenous genes that encode L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4-epimerase, transaldolase and transketolase which impart arabinose to ethanol fermentation capability. The plasmid vector (pZB 206) and a process of using the transformants of the fermentation of a glucose and arabinose containing substrate is also described.
U.S. Pat. No. 5,843,760 discloses a Z. mobilis xylose and arabinose fermenting transformant (206C/pZB301) containing exogenous genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase and transketolase, and further comprising at least one promoter recognized by Zymomonas which regulates the expression of at least one of said genes, wherein said microorganism is capable of growing on arabinose and/or xylose, alone or in combination, as the carbon source and fermenting said arabinose and xylose to ethanol. The process of using the transformants together with the plasmid vectors (pZB301, pZB401, pZB402, and pZB 403) is also described.
We have established that the production of bioethanol by Z. mobilis is regulated by the quorum sensing (QS) molecule AI-2 (Bassler B L et al. (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J. Bacteriol. 179, 40-3, Chen, X et al. (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415, 545-9). The use of AI-2 to enhance bioethanol production is likely to be a better tactic than simply engineering bacteria, since one can expect that the expression of all the genes necessary for enhancing production will be regulated in a coordinated manner, whilst also activating mechanisms to increase the tolerance of the bacterium to the increased levels of ethanol.